1. Field of the Invention
The present invention relates to a preparation of an optically active substance having a physiological activity. More specifically, it relates to an enzymatic preparation of an intermediate for synthesizing an optically active substance (e.g., epihalohydrin), i.e., a so-called "chiral synthon", capable of reducing the synthesis steps of the optically active substance. The present invention also relates to a bioreactor system suitable for use in an enzymatic preparation of an optically active substance useful as an important intermediate for synthesis of, for example, pharmaceutical products using a powdered or granulate enzyme in an organic solvent.
2. Description of the Related Art
Optically active pharmaceutical products have been heretofore produced by fermentation (enzyme) methods using microorganisms (enzymes), pure organic synthesis methods, or combination of the above-mentioned two methods. Of these methods, when non-natural type pharmaceutical products are produced, the organic synthesis methods or the combined methods are generally used. According to the organic synthesis methods, optically active pharmaceutical products having structures completely different from those of natural products can be prepared and, according to the combined methods, optically active pharmaceutical products having structures similar to those of natural products can be prepared.
The above-mentioned organic synthesis methods can advantageously prepare new types of pharmaceutical products having completely different new structures. However, there are disadvantages in these methods that the starting materials are relatively expensive optically active natural sugars or amino acids and that the synthesis steps or routes are generally long.
On the other hand, recently, effective syntheses of optically active synthetic intermediates (i.e., chiral synthons) to reduce the synthesis steps or routes of the organic synthesis methods are noted. Of these chiral synthon, .beta.-blocker synthesis intermediates such as (R)-solketal and optically active epoxides are commercially available. For example, ##STR1## However, according to these methods, the optically active intermediates are only derived from optically active natural products such as sugars and amino acids and new synthesis methods for chiral synthon have not been developed.
Recently, Iriuchijima, Hamaguchi et. al. reported an effective enzymatic synthesis of glycerol derivatives, which are chiral synthon of .beta.-blocker, i.e., an arrhythmia agent in Agric. Biol. Chem., 46, 1153 (1982) and Agric. Biol. Chem., 50, 1629 (1986). However, since enzymes dissolved in water are used in these experiments, it is difficult to recover and reutilize these enzymes. On the other hand, A. M. Klibanov reported the preparation of optically active esters or alcohols in J. Am. Chem. Soc., 107, 7072 (1985) by using a racemic alcohol as a starting material, a solid enzyme suspended in an organic solvent, and an esterified alcohol. However, it is reported that, in this method, a secondary alcohol is used as a racemic alcohol and that, when a primary alcohol is used, the desired product having a high optical purity cannot be obtained and no specific examples are given in this reference.
Furthermore, the following methods are reported as the synthesis methods of optically active epihalohydrins.
1 Synthesis from D-mannitol (J. J. Baldwin, J. Org. Chem., 43, 4876, (1978))
According to this method, (S)- and (R)-epichlorohydrins are synthesized from D-mannitol. However, this method is disadvantageous in that the synthesis route is long and the use of heavy metal compounds such as lead tetraacetate causes safety problems.
2 Synthesis from optically active 2,3-dichloro-1-propanol (JP-A-62-6697)
(R)-2,3-dichloro-1-propanol is removed from the racemic mixture thereof by utilizing the same with an immobilized microorganism and the resultant (S)-2,3-dichloro-1-propanol is treated with a two-layer solution of ether/aqueous NaOH solution, followed by cyclization to obtain (R)-epichlorohydrin. However, this method is disadvantageous in that a cumbersome immobilizing treatment of a microorganism, sterilization of the reaction apparatus and the like are required and that the synthesis of (S)-epichlorohydrin is impossible.
3 A method in which starting materials and enzymes similar to those of the present invention are used (Shigeki Hamaguchi; Text of the 22nd Biochemical Engineering Lecture Course, page 41, (1987))
Although this method proposes the use of starting materials and enzymes similar to those of the present invention, the recovery and reutilization of enzymes and the attainment of the continuous reaction are impossible because of the aqueous reaction, and because cumbersome post-treatments such as extraction are required. Furthermore, during the enzymatic hydrolysis reaction, free acids are generated and cause non-enzymatic (i.e., non-stereoselective) hydrolysis. Thus, since the optical purity of the desired products, (S)-alcohols or (R)-esters is decreased, the reaction must be carried out while the generating acid is neutralized with an aqueous NaOH solution. Furthermore, since the starting racemic esters have a low solubility in water, the increase in the reaction temperature and the addition of an organic solvent are required to accelerate the hydrolysis reaction. These actions, however, can deactivate the enzymes.
In addition, after the enzymatic hydrolysis reaction, although the desired (R)-esters and (S)-alcohols can be obtained by extraction, recovery and reutilization of enzymes used is extremely difficult because of the aqueous reaction.
Moreover, according to this method, the resultant mixture of (R)-ester and (S)-alcohol is directly treated, without separation, with an aqueous NaOH solution having a pH of 12, whereby only the (S)-alcohol is cyclized to form (S)-epichlorohydrin and the (R)-ester is recovered. The recovery of (R)-ester and (S)-epichlorohydrin should be carried out by extraction.
Heretofore, various methods for obtaining optically active substance have been known. Examples of such methods are (a) optical resolution of racemic mixtures by enzymes and microorganisms, (b) optical resolution of racemic mixtures by chemical methods, (c) methods for deriving optically active substances from optically active natural products, (d) asymmetric syntheses by enzymes and microorganisms, and (e) asymmetric syntheses by chemical methods. Of these methods, the methods (a) and (d) using biocatalysts such as enzymes and microorganisms have generally become noted as the best methods for practical use because of mild reaction conditions and a high selectivity.
For example, the above-mentioned method (a) using a bioreactor is commercially utilized in the production of optically active amino acids, i.e., racemic acylamino acids are hydrolyzed by reaction kinetics optical resolution using enzymes, whereby the enantiomer is optically resoluted. According to this method, immobilized enzymes bonded to DEAE-Sephadex by a carrier bonding method are used. However, since this reaction is carried out in an aqueous medium, a problem arises in that hydrophobic substrates are not easy to treat (Chibata et. al., Agr. Biol. Chem., 33 (7), 1047-1052, 1969). Furthermore, this method has a problem in that the retentionability of enzyme activity is low. For example, the enzyme activity in a fixed bed type reactor is decreased by 60% in 32 days.
The above-mentioned method (d) is also practically used in the commercial production of L-asparginic acid or L-malic acid in a bioreactor using an immobilized enzyme. This reaction is also carried out in an aqueous medium (Chibata et. al., Appl. Microbiol. 27, 878, 1974). Furthermore, since this method uses, as an immobilization method, a gel-entrapment immobilization method in which polyacrylamide or copper carageenan is used, the immobilization operation is complicated and, since the reaction is carried out in an aqueous medium, the reaction of hydrophobic substrates is difficult.
Furthermore, a research report of the above-mentioned method (a) using both water and organic solvents has been proposed in Asada et. al., JP-A-60-78596. According to this method, racemic compounds are optically resolved by hydrolases. However, this method has disadvantages from the practical viewpoint in that a complicated immobilization treatment of enzymes is required and that, since water and an organic solvent must be alternately used, the operations are complicated and the process control is difficult.
Recently, it has been recognized in the art that an organic solvent type bioreactor capable of increasing the reactivity by solubilizing hydrophobic substrates is necessary. Various studies have been made to develop such a bioreactor. For example, methods using immobilized enzymes (i.e., ion bonding method+crosslinking method) (see Matsuno et. al., Bio/Technology, 3, 459, 1985) or methods for separating enzymes from hydrophobic products and for effecting the reactions at the interfaces of micro-pores of thin membranes by using micropore thin membranes (e.g., precision filter membranes) (see Yamane et. al., Yu Kagaku, 33, 683, 1984).
According to Matsuno et. al. method, the enzymes are likely to be deactivated when immobilized, especially when crosslinked with glutaric aldehyde and, furthermore, extensive work and time are required for the immobilization operation. On the other hand, according to Yamane et. al. method, the reactivity largely depends upon the water content of glycerol solution. For example, the maximum reactivity can be obtained at the water content of 3 to 4% and the reactivity is decreased either below or above this water content. In addition, according to this ester synthesis reaction, since water is always formed, it is very difficult to maintain the water content in the system and, furthermore, complicated reactors are required. Thus, this method is not suitable for practical use.
As bioreactors for enzymatic reactions, two types, i.e., homogeneous phase type bioreactors in which enzymes are solubilized in aqueous media and two phase type bioreactors in which enzymes are used in the immobilized state, have been heretofore used. Of these bioreactors, the homogeneous bioreactors include, for example, agitating vessel type bioreactors and ultrafiltration membrane type reactors. However, these bioreactors, especially except for those using ultrafiltration membrane, have disadvantages in that, since catalytic enzymes flow out from the vessels, the continuous reaction advantageous for the commercialization cannot be readily realized.
The two-phase type bioreactors generally use immobilized enzymes and include, for example, the agitating vessel type, fixed or packed bed type, fluidized bed type, membrane type, and suspension air bubbling column type reactors, all of which can be used in a continuation reaction system.
As catalysts for bioreactors, various kinds of substances other than purified enzymes have been recently used. That is, in the case of, for example, intracellular enzymes, which are difficult to isolate, "treated cells" obtained by treating the same by heat, organic solvents, or surfactants to retain only the property capable of catalyzing the desired reaction can be used. "Pausing or resting cells", which are living cells but do not proliferate or "proliferating cells" capable of proliferating in the reactor, can also be used. These enzymes can be used not only in a one step reaction but also in a multi-step reaction.
As immobilization methods for insolubilizing enzymes in water, various methods including a carrier bonding method, a crosslinking method, and a gel-entrapment method are known. However, these methods generally require a complicated treating method and enzymes sometimes are deactivated to cause a decrease in the activity of the enzymes. In the case of the gel-entrapment methods and the like, the permeability of substrates and products into polymeric substances surrounding the enzymes is low and, therefore, the reactivity is unpreferably decreased. On the other hand, in the case of the physical adsorption methods (carrier bonding methods), in which the enzymes are adsorbed on the surface of inorganic carriers or polymeric substances, the interaction between the enzyme and the carrier is weak and, therefore, the adsorbed enzyme is sometimes eliminated from the surface of the carrier. Thus, these methods are disadvantageous for the continuous reaction in an aqueous solution.
Furthermore, regarding the reaction solvents, the use of an organic solvent has gradually increased, in addition to the conventional aqueous solution, and it is recognized that (immobilized) enzymes microorganisms are relatively stable even in organic solvents. Examples of these proposals are the use of powdered enzymes in organic solvents for a production of diglycerides from the ester synthesis reaction of glycerol and fatty acids (JP-A-62-19090) and the use of powdered enzymes in organic solvents for synthesis of optically active substances in batchwise reactions (Klibanov et. al., J. Am. Chem. Soc., 107, 7072, 1985). However, although the enzymes can be recovered by filtration and can be repeatedly used, there are various problems in the practical use thereof in commercial production because of, for example, the cumbersome filtration operation and fluctuations in the reactivity caused by a difficulty in control of the water content in the enzymes when repeatedly used.